The overall objective of the proposed research is to investigate the initiation of protein biosynthesis in chloroplasts. Two initiation factors required for this process have been purified from the chloroplasts of Euglena gracilis. The factor designated IF-2chl is required for binding the initiator tRNA to the small ribosomal subunit. It is structurally complex and appears in several high molecular weight forms. In the current proposal, we plan to investigate the relationships between these forms using a variety of techniques including probing with monoclonal antibodies. A detailed analysis of the mechanism of action of this factor will be carried out. In addition, we plan to clone and sequence the nuclear gene for this factor. The second initiation factor (IF-3chl) appears to promote the dissociation of ribosomes and stimulates initiation complex formation on chloroplast 30 S ribosomal subunits. We will examine structure and mechanism of IF-3chl and will seek to isolate and characterize the nuclear gene for this factor. Our third aim is to investigate the nucleotide sequences that are involved in the formation of initiation complexes in the chloroplasts of E. gracilis. The mRNAs in this organelle appear to fall into two classes - those that have a chloroplast sequence equivalent to the Shine/Dalgarno sequence found in prokaryotic mRNAs and those that lack a Shine/Dalgarno sequence. We will use deletion and site-directed mutagenesis to investigate the sequences that are important for initiation on these two classes of messages. Finally, we will extend our studies of chloroplast initiation to a higher plant system. We will seek to develop an in vitro system for the formation of translation initiation complexes on tobacco chloroplast 30 S subunits and will examine the nucleotide sequences important for initiation complex formation in this system. Our long term goal is to develop a clear understanding of the mechanism of initiation of protein synthesis in photosynthetic organelles and to lay the foundation for its regulation and its integration into the complex metabolism of the cell.